Mapping DNA polymerase errors by single-molecule sequencing.
Nucleic Acids Res
; 44(13): e118, 2016 07 27.
Article
em En
| MEDLINE
| ID: mdl-27185891
ABSTRACT
Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replication product is tagged with a unique nucleotide sequence before amplification. This allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA Polimerase Dirigida por DNA
/
Replicação do DNA
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Sequenciamento de Nucleotídeos em Larga Escala
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Estados Unidos